CN114478779A - Humanized phosphatidylinositolglycan 3 monoclonal antibody and application thereof - Google Patents

Humanized phosphatidylinositolglycan 3 monoclonal antibody and application thereof Download PDF

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CN114478779A
CN114478779A CN202011158008.2A CN202011158008A CN114478779A CN 114478779 A CN114478779 A CN 114478779A CN 202011158008 A CN202011158008 A CN 202011158008A CN 114478779 A CN114478779 A CN 114478779A
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丰明乾
李静文
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Huazhong Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]

Abstract

The present invention provides high affinity humanized monoclonal antibodies that bind GPC3 protein. The humanized monoclonal antibody retains the affinity similar to that of the original monoclonal antibody, has lower immunogenicity, and may be used in developing GPC3 antibody medicine, antibody-medicine conjugate or immunotoxin medicine with high activity.

Description

Humanized phosphatidylinositolglycan 3 monoclonal antibody and application thereof
Technical Field
The present invention relates to the field of antibodies. In particular to a humanized phosphatidylinositol proteoglycan 3 monoclonal antibody.
Background
Today, antibodies are widely used substances in medicine and research. In medicine, they find application in many different fields. For example, antibodies are used as labeling reagents for detecting certain markers, which allows diagnosis or prognosis of a disease or determination of a particular physical parameter. For example, the detection of protein molecules on the surface of certain cancer cells.
In addition, the antibodies are also useful as therapeutic agents in the treatment and prevention of various diseases, such as cancer, cardiovascular diseases, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infections. In these treatments, the antibodies themselves may have therapeutic activity, for example by blocking receptor or messenger molecules, thereby inhibiting their disease-related functions, or by restoring and activating components of the patient's immune system. Alternatively, the antibody may be conjugated to another substance having therapeutic activity. In particular, in the treatment of cancer and infections, the other substance has cell killing activity and may be, for example, a radioisotope or a cytotoxin. In another application, the antibodies can be used to passively immunize a patient by transferring the appropriate antibody into the patient's circulation.
Currently, hybridoma technology and phage display technology are commonly used for preparing monoclonal antibodies, and antibodies aiming at single specific epitopes of antigens can be generated. These antibody sequences are often derived from mammals, such as mice and rabbits. From an evolutionary point of view, mammalian relatives are very close to humans, and many proteins are homologous, so that corresponding antibodies are produced in these animals.
The chicken relativity is farther away from human, and the immune chicken can generate antibodies covering more epitopes. The sequence of the framework region of the chicken antibody is only one, and a phage display library is easier to construct.
However, in therapeutic applications, these monoclonal antibodies have the problem of being derived from animal organisms and differing in their amino acid sequence from human antibodies. Thus, the human immune system recognizes these animal antibodies as foreign substances and rapidly clears them from the circulation. In addition, systemic inflammatory effects may result. Therefore, antibodies of animal origin need to be further humanized.
One common method of antibody humanization is complementarity determining region grafting, in which CDRs from a non-human antibody are grafted onto a human framework. Generally, the human framework with the highest homology to the framework regions of the non-human antibody is selected as the recipient for CDR grafting. During humanization of monoclonal antibodies, the affinity and specificity of the engineered antibody is often reduced. In practical applications, the tight binding and specific recognition of the antigen to the antibody used is crucial. Therefore, it is very significant to select the optimal framework sequence, which can make the humanization degree higher and can not lose the activity of the antibody.
GPC3(Glypican-3) is a member of heparin sulfate proteoglycans, anchored to the surface of cell membranes by glycosyl-phosphatidylinositol (GPI). The human GPC3 gene is located on the X chromosome (Xp26) and encodes a 70kDa protein, containing 580 amino acids, which is cleaved endonucleases between Arg358 and Ser359 with a furin-like convertase to yield a 40kDa N-terminal subunit and a 30kDa C-terminal subunit, with two Heparan Sulfate (HS) chains on the C-terminal subunit. Studies have shown that GPC3 is expressed at a significantly up-regulated level in approximately 72% hepatocellular carcinoma (HCC) compared to normal hepatocytes, cholangiocarcinomas and liver metastases, but not in liver tissue of normal adults. In addition, high expression of GPC3 in liver cancer patients is often associated with a poor prognosis, which indicates the potential role of GPC3 as a biomarker in HCC. GPC3 has been proposed as a target for antibody and cell-based immunotherapy.
Disclosure of Invention
According to the early research, after chicken is immunized by GPC3-hFc protein, an antibody is obtained by screening through a phage display technology, then the antibody is preliminarily classified according to epitopes through phage sandwich ELISA, and a series of antibodies which can recognize different epitopes of glypican 3 with high affinity and have good stability are screened through an antibody protein sandwich ELISA method. Based on the invention, after most sequences in the framework region of the GPC3 high-affinity chicken antibody are replaced by the adult framework region, the humanized antibody not only has reduced immunogenicity, but also retains the high-affinity characteristic.
The specific technical scheme of the invention is as follows:
the present invention is based on the generation of humanized anti-GPC 3 antibodies with antigen binding properties similar to the corresponding chicken antibodies.
The invention provides a humanized monoclonal antibody of glypican 3, the humanized antibody comprises a heavy chain variable region and a light chain variable region, and the framework region of the heavy chain variable region: the HFR1 amino acid sequence is shown as 1-25 of SEQ ID NO. 3, the HFR2 amino acid sequence is shown as 35-49 of SEQ ID NO. 3 or 35-49 of SEQ ID NO. 19, the HFR3 amino acid sequence is shown as 67-98 of SEQ ID NO. 3, 67-98 of SEQ ID NO. 15, or any one of 67-98 of SEQ ID NO. 17, the HFR4 amino acid sequence is shown as 116-126 of SEQ ID NO. 3, and the framework region of the light chain variable region is: the LFR1 amino acid sequence is shown as 1-22 bits of SEQ ID NO. 4, the LFR2 amino acid sequence is shown as 30-45 bits of SEQ ID NO. 4 or 30-45 bits of SEQ ID NO. 14, the LFR3 amino acid sequence is shown as 53-84 bits of SEQ ID NO. 4, 53-84 bits of SEQ ID NO. 6, 53-84 bits of SEQ ID NO. 8, 53-84 bits of SEQ ID NO. 10, or any one of 53-84 bits of SEQ ID NO. 12, and the LFR4 amino acid sequence is shown as 98-107 bits of SEQ ID NO. 4.
Further, the humanized antibody is preferably selected from the group consisting of:
(1) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 3; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO. 4;
(2) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 5; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 6;
(3) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO. 7; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 8;
(4) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 9; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 10;
(5) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 11; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 12;
(6) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 13; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO. 14;
(7) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 15; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 16;
(8) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 17; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 18;
(9) the heavy chain variable region of the humanized antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 19; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO: 20;
in a specific technical scheme of the invention, the humanized antibody of glypican 3 comprises a heavy chain variable region and a light chain variable region,
(1) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 3, and the light chain variable amino acid sequence is shown as SEQ ID NO. 4.
(2) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO.5, and the light chain variable amino acid sequence is shown as SEQ ID NO. 6.
(3) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 7, and the light chain variable amino acid sequence is shown as SEQ ID NO. 8.
(4) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 9, and the light chain variable amino acid sequence is shown as SEQ ID NO. 10.
(5) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 11, and the light chain variable amino acid sequence is shown as SEQ ID NO. 12.
(6) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 13, and the light chain variable amino acid sequence is shown as SEQ ID NO. 14.
(7) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 15, and the light chain variable amino acid sequence is shown as SEQ ID NO. 16.
(8) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 17, and the light chain variable amino acid sequence is shown as SEQ ID NO. 18.
(9) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 19, and the light chain variable amino acid sequence is shown as SEQ ID NO. 20.
In a specific embodiment of the present invention, the heavy chain variable region of the humanized antibody has complementarity determining regions represented by amino acid residues 26-34, 50-66 and 99-115 of SEQ ID NO. 3; the variable region of the light chain of the antibody has complementarity determining regions shown by amino acid residues 23-29, 46-82 and 85-97 of SEQ ID NO. 4.
The monoclonal antibodies of the invention can be of any isotype. May be, for example, an IgM or IgG antibody, such as IgG1 or IgG 2. The classes of antibodies that can specifically bind GPC3 can be converted from each other according to known methods (e.g., IgG can be converted to IgM). Class switching can also be used to switch one IgG subclass to another, for example from IgG1 to IgG 2.
The antibody of the invention may be:
(1) fab, a fragment comprising a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of an intact antibody with papain to produce an intact light chain and a portion of one heavy chain;
(2) fab' antibody molecule fragments obtainable by treatment of an intact antibody with pepsin followed by reduction to produce a portion of the intact light and heavy chains; two Fab' fragments were obtained per antibody molecule;
(3) (Fab')2, an antibody fragment obtainable by treating an intact antibody with pepsin, but without subsequent reduction; f (ab ')2 is a dimer of two Fab' fragments joined together by two disulfide bonds;
(4) fv, a gene-engineered fragment containing the variable regions of the light and heavy chains expressed as 2 chains;
(5) single chain antibodies (e.g., scFv), genetically engineered molecules that contain a light chain variable region and a heavy chain variable region and are linked by a suitable polypeptide linker into genetically fused single chain molecules;
(6) dimers of single chain antibodies (scFv2), defined as dimers of scFv (also known as "minibodies");
(7) a VH single domain antibody, an antibody fragment consisting of the variable region of the heavy chain.
One skilled in the art will appreciate that conservative variants of the antibody may be made. Amino acid substitutions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) may be made in the VH and/or VL regions, with the substituted VH and VL still retaining the ability to bind GPC3, or being more capable of binding GPC 3. Conservative substitutions of functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered conservative substitutions for one another:
1) alanine (a), serine (S), threonine (T);
2) aspartic acid (D), glutamic acid (E);
3) asparagine (N), glutamine (Q);
4) arginine (R), lysine (K);
5) isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenylalanine (F), tyrosine (Y), tryptophan (W).
The humanized antibody or a fragment or derivative thereof according to the invention is preferably used in medicine, in particular in the treatment, diagnosis, prognosis and monitoring of diseases, in particular of diseases described herein, preferably cancer.
The GPC3 antibody of the present invention can be conjugated to an effector molecule. Effector molecules include, but are not limited to, toxins, drugs, or detectable labels.
The drug of the present invention is a substance having cytotoxic or antitumor activity, such as Monomethylauristatin E (MMAE), Monomethylauristatin F (MMAF), Pyrolobe diazepine (PBD) dimer, N2'-deacetyl-N2' - (3-Mercapto-1-oxopropyl) -Maytansine (Maytansine DM1), vinblastine, daunomycin and the like, andsuch as radioactive agents125I、32P、14C、3H and35s and the like.
The toxin is a toxic protein with cytotoxicity or antitumor activity, and can be conjugated with the antibody to form immunotoxin, including but not limited to pseudomonas exotoxin, ricin, abrin, diphtheria toxin and subunits thereof, and botulinum toxin A-F, and truncated mutants and point mutants of the toxins. These toxins are commercially available (e.g., Sigma Chemical Company, st. louis, MO). The toxins also include variants of the above toxins (see, e.g., U.S. patent nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas Exotoxin (PE) (U.S. Pat. No.5,602,095). The "pseudomonas exotoxin" includes its native sequence, a cytotoxic fragment of the native sequence, and conservatively modified variants of the native sequence or a cytotoxic fragment thereof. These modifications include, but are not limited to, the removal of multiple amino acid deletions in domains Ia, Ib, II and III, single or multiple amino acid substitutions and the addition of one or more sequences at the carboxy terminus (see, e.g., Siegel et al, J.biol. chem.264: 14256-14261, 1989). Cytotoxic fragments of pseudomonas exotoxin include PE24, PE40, PE38, PE35, and the like.
The detectable label of the present invention is a substance that can be detected by an isotope analyzer, a microplate reader, a bioluminescent detector, a chemiluminescent detector, an electrochemiluminescent detector, a fluorescent analyzer, or visualized by the naked eye, including, but not limited to, radioisotopes (e.g., radioisotopes)3H、14C、15N、35S、90Y、、99Tc、111In、125I、131I) Enzymes (such as horseradish peroxidase, beta-galactosidase, alkaline phosphatase, glucose oxidase and the like) which can be used for detection, fluorescent proteins (such as Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), allophycocyanin APC, phycoerythrin PE), bioluminescent markers (such as luciferase), fluorescent compounds (such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethyl-5-D-methyl-L-beta-galactosidase, etc.), fluorescent proteins (such as beta-galactosidase, beta-galactosidase, etc.), and the likeAmino-1-naphthalenesulfonyl chloride, phycoerythrin, fluorescent dyes Cy3, Cy5, rare earth phosphors, quantum dots, etc.), biotin, a magnetic reagent (e.g., gadolinium), an electrochemiluminescent reagent (e.g., ruthenium terpyridyl), and colloidal gold.
It is another object of the present invention to provide a polynucleotide encoding the antibody, recombinant protein or immunoconjugate of the present invention. Another objective of the invention is to provide a vector containing the polynucleotide of the invention. The carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
The present invention provides a host cell comprising a nucleic acid according to the invention or an expression cassette or vector according to the invention. The host cell according to the invention may be any host cell. May be isolated cells or cells contained in a tissue. Preferably, the host cell is a cultured cell, in particular a primary cell or a cell of an established cell line, preferably a human cell derived from a tumor.
Another objective of the present invention is to provide a pharmaceutical composition, which comprises one or more of the antibodies, recombinant proteins, immunoconjugates, polynucleotides, vectors or genetically engineered host cells described in the present invention. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The antibody, recombinant protein, immunoconjugate, polynucleotide, vector or genetically engineered host cell may be soluble in an aqueous carrier, such as buffered saline or the like. May also contain pharmaceutically acceptable adjuvants required to approximate physiological conditions, such as pH regulator and buffer, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
Another objective of the invention is to provide an application of the antibody, the recombinant protein, the immunoconjugate, the polynucleotide, the vector or the genetically engineered host cell in preparing a therapeutic drug or a diagnostic reagent for autoimmune diseases, viral infections or cancers. The cancer is liver cancer, stomach cancer, colorectal cancer, lung cancer or ovarian cancer, or any other type of cancer expressing GPC 3.
The monoclonal antibodies disclosed herein can also be used to make chimeric antigen receptors (CAR; also known as chimeric T cell receptors, artificial T cell receptors, or chimeric immunoreceptors) or bispecific antibodies.
The antibodies and compositions provided by the present invention can be used for a variety of purposes, such as molecular diagnostics of tumors, confirming the expression of GPC3 in liver cancer and other tumor patient samples. The sample may be any sample, including but not limited to tissue from biopsies, autopsies, and pathological specimens. Biological samples also include sections of tissue, such as frozen sections taken for histological purposes. Biological samples also include bodily fluids such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are typically obtained from mammals, including humans, non-human primates, mice, and the like.
The invention has the advantages that: the invention provides a humanization strategy of a GPC3 chicken monoclonal antibody. In this example, the variable regions of the heavy and light chains (VH, SEQ ID NO:21 and VL, SEQ ID NO:22) of the chicken monoclonal antibody A5, GPC3, and A5 were linked with linker peptide G4S to form the scFv structure. Based on the structure of the scFv, the most relevant framework regions were selected from the human germline antibody library according to their overall sequence similarity and CDR loop classification: framework regions 1, 2 and 3 of the heavy chain variable region were selected from human germline VH gene 3-23 x 02. Framework region 4 of the heavy chain variable region was selected from human germline gene J3 x 01. Framework regions 1, 2 and 3 of the light chain variable region were derived from human germline VL gene 3-25 x 02. Framework region 4 of the light chain variable region was selected from human germline gene J7 x 01 and optimized to give the humanized antibody with desirable affinity and lower immunogenicity. Can be used for developing GPC3 antibody drugs, antibody-drug conjugates or immunotoxin drugs with better activity.
Drawings
FIG. 1FACS method the binding activity of antibody A5 prepared in example 1 of the present invention to GPC3 negative/positive cell line was examined.
FIG. 2 alignment of the framework regions and complementarity determining regions of the heavy chain variable region of the humanized chicken antibody of example 4 of the present invention.
FIG. 3 alignment of the framework region and the complementarity determining region of the light chain variable region of the humanized chicken antibody of example 4 of the present invention.
FIG. 4 SDS-PAGE analysis of the purity of HuA5.2 antibody and 8 mutant proteins.
Figure 5 ELISA detects the affinity of the hua5.2 antibody and 8 mutants to bind to the antigen GPC 3.
FIG. 6 SDS-PAGE detection of purity of HuA5.2 antibody purified from eukaryotic expression System
FIG. 7 ELISA for detecting the affinity of the binding of HuA5.2 and A5 antibodies to antigen
FIG. 8 ELIspot assay for immunogenicity of HuA5.2 and A5 antibodies
Detailed Description
The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1 preparation of chicken monoclonal antibody A5 that binds GPC3
Immunizing 4-month-old chicken with GPC3-hFc protein (Sino Biological, Cat:10088-H02H2), immunizing 300 μ g each time, 2 weeks apart each time, collecting spleen of chicken after immunizing 4 times, extracting total RNA in spleen, reverse transcribing into cDNA, using the cDNA as template for establishing phage antibody library, and constructing chicken antibody phage library (library capacity 2.7 × 10)9). After 4 panning of the phage library using the GPC3 protein as the antigen, 300 clones were randomly picked and sequenced, and the sequences were aligned to find that a5 was the most frequent antibody.
The scFv of A5 was fused with hFc to construct the expression vector pPBSPS-A5-hFc, which was expressed in 293F cells. The expression product was purified by protein A chromatography (GE healthcare). GPC3 protein was detected using purified antibodies and sandwich ELISA.
The VH of A5 is shown in SEQ ID NO:21, the VL is shown in SEQ ID NO:22, and the CDR and HFR region sequences are respectively shown in Table 1.
TABLE 1 amino acid sequence of the variable region of the GPC3 Chicken monoclonal antibody
Figure BDA0002743374460000081
Example 2 in vitro characterization of chicken monoclonal antibodies that bind GPC3
SPR measurement of binding kinetics and affinity of the antibody prepared in example 1 to GPC3
The GPC3-his protein was immobilized on a carboxymethyl sensor chip (S series sensor chip CM5) by standard amine coupling. The chip was washed to obtain a stable baseline, and then different concentrations of antibody analyte and running buffer were injected into the chip at a flow rate of 30 μ L/min with a sample binding time of 180 seconds followed by a dissociation time of 600 seconds. Binding and dissociation curves were fitted to a 1:1Langmiur binding model using ProteOn software. The results show that the affinity constant Kd value of A5 is 1.82E-11, indicating that its affinity for GPC3 is very high.
3. Detection of antibody stability
The stability of A5 was determined using Prometheus NT.48 from Nano tester: the antibody concentration was diluted to 50ug/ml and then loaded, the temperature rose from 25 ℃ to 95 ℃ within 40min at a rate of 1.5 ℃/min. And finally obtaining the Tm value of the antibody. The results showed that A5 had a Tm of 69.1 ℃ indicating that A5 has good thermal stability.
Example 3FACS detection of binding of the antibodies of the invention to a GPC3 positive cell line
A431, G1 (A431 cell line overexpressing GPC 3) (Phung Yet. mabs 2012; 4: 592. 599) and liver cancer cell lines HepG2, Hep3B, HuH-7 were cultured adherently in DMEM medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% L-glutamine and 1% penicillin-streptomycin (Invitrogen, Carlsbad, Calif.). After harvesting the cells, the antibodies prepared in example 1 were bound to a431, G1, HepG2, Hep3B, Huh7 cells, respectively, and APC-labeled goat anti-human secondary antibodies were added to detect the antibodies bound to the cell surface. As shown in FIG. 1, the antibody prepared in example 1 strongly bound to G1 cells and HepG2, Hep3B and Huh7 cells, but did not bind to GPC 3-negative A431 cells, indicating that the antibody prepared in example 1 specifically recognizes and binds to the GPC3 protein on the cell surface.
Example 4 humanization of the chicken heavy and light chain variable regions of an anti-GPC 3 antibody
In this example, the A5 antibody was used as an example, and the variable regions of its heavy and light chains (VH, SEQ ID NO:21 and VL, SEQ ID NO:22) were linked by the linker peptide G4S to form the scFv structure. Based on the structure of the scFv, the most relevant framework regions were selected from the human germline antibody library according to their overall sequence similarity and CDR loop classification: framework regions 1, 2 and 3 of the heavy chain variable region were selected from human germline VH gene 3-23 x 02. Framework region 4 of the heavy chain variable region was selected from human germline gene J3 x 01. Framework regions 1, 2 and 3 of the light chain variable region were derived from human germline VL gene 3-25 x 02. Framework region 4 of the light chain variable region was selected from human germline gene J7 x 01.
The first humanization scheme is as follows: the framework regions 1, 2, 3 and 4 of the chicken heavy chain variable region and the light chain variable region are all replaced by humanized framework regions, and the heavy chain variable region of the antibody has the framework regions shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 1; the variable region of the light chain of the antibody has framework regions shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 2. The antibody sequence obtained in this protocol was designated HuA5.1.
And B, a second humanization scheme: according to the literature data (Leeying Wu, J Immunol 2012; 188:322-333), the positions with high mutation rates in the framework regions 1, 2, 3 and 4 of the chicken heavy chain variable region and the chicken light chain variable region are replaced by the amino acids at the corresponding positions of the humanized framework region, and the heavy chain variable region of the antibody has the framework regions shown by the amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 3; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO. 4; . The antibody sequence obtained in this protocol was designated HuA5.2.
A third humanization scheme: on the basis of the second humanization scheme, the mutation rate in the framework regions 1, 2, 3 and 4 of the chicken heavy chain variable region and the chicken light chain variable region is low, namely, the sequences of the chicken which are conserved are replaced by the sequences of the adult one by one. The 35 th G, 67 th A and 78 th V variable regions of the HuA5.2 heavy chain are mutated to S, F and L respectively by reference to Kabat and IMGT numbering rules. The variable region of the HuA5.2 light chain was changed from 36 th F to Y, 57 th D to G, 60 th S to D,66 th A to S, and 69 th S to N, respectively. The antibody sequences obtained by the scheme are named as H-G35S, H-A67F, H-V78L, L-F36Y, L-D57G, L-S60D, L-A66S and L-S69N respectively.
In total, 10 humanized antibodies of different sequences were obtained. The antibody heavy and light chain sequences are shown in table 2, and the framework regions of the variable regions are shown in table 3. The difference in sequence alignment between the framework region and the complementarity determining region in the antibody heavy chain variable region is shown in FIG. 2. The difference in sequence alignment between the framework region and the complementarity determining region in the antibody light chain variable region is shown in FIG. 3.
TABLE 2.10 variable region amino acid sequences of humanized antibodies
Figure BDA0002743374460000091
Figure BDA0002743374460000101
TABLE 3.10 FR amino acid sequences of humanized antibodies (according to Kabat and IMTG databases)
Figure BDA0002743374460000102
Figure BDA0002743374460000111
Example 5 the humanized antibody of example 4 was expressed and tested for purity
The nucleic acid fragment corresponding to the antibody in example 1 was synthesized by Kinsley, and then 6 histidines (6 XHis) were added to the C-terminal sequence of the antibody to construct a pET28a expression vector for the corresponding antibody, followed by transformationBL21 E.coli, inoculating the strain into 2L 2YT medium, culturing at 37 deg.C for 4-5h, and culturing to obtain OD600Reaching 0.9, adding 1mM IPTG for induction, and expressing for 10h at 30 ℃. Collecting thallus, crushing thallus under high pressure, centrifuging, filtering to obtain supernatant, and purifying with nickel column. Among them, the HuA5.1 antibody could not be expressed, and the rest antibodies could be expressed and purified. The results show that after the framework regions are completely replaced by the human framework, the conformation of the antibody is greatly changed, and finally the antibody cannot be purified. And finally, detecting the purity of the rest purified protein by SDS-PAGE. The size of the antibody prepared in example 4 is about 50Kd, and as shown in FIG. 4, the band size is correct, wherein the purity of 4 antibodies is higher, and the purity of the remaining 5 antibodies is general, but the requirements of the subsequent experiments are met.
Example 6ELISA assay of the Activity of the humanized antibody prepared in example 5 to bind GPC3
GPC3 protein was coated on the bottom of an ELISA plate, and the antibody proteins in example 4 were then separately started at 20 μ g/ml 1: 3, adding the mixture into a hole after gradient dilution, then adding an HRP-mouse anti His antibody, and finally adding a substrate for color development. As shown in FIG. 5, all antibodies HuA5.2, H-G35S, H-A67F, H-V78L, L-F36Y, L-D57G, L-S60D, L-A66S, and L-S69N were able to bind significantly to the GPC3 antibody. The affinity constant Kd value of the antibody was calculated from the results of ELISA, as shown in Table 4, in which the affinity of HuA5.2 was the highest and the Kd value reached 0.02051. The results show minimal loss of affinity for hua5.2 after humanization.
Table 4 affinity EC50 values for binding of the antibodies in example 5 to GPC3 protein
Figure BDA0002743374460000121
Example 7293 cells express HuA5.2 protein and purity is tested
Considering the subsequent detection of the immunogenicity of the HuA5.2 antibody, the HuA5.2 is expressed in a mammalian cell expression system, and the additional introduction of immunogen by an Escherichia coli expression system is avoided. Adding an additional hFc tag to the C end of the sequence of HuA5.2 to construct a mammalian cell expression vector, then transiently transferring the successfully constructed vector to 293F cells, collecting cell supernatant after 7 days of suspension culture, filtering and purifying by a ProteinA chromatographic column. The purity was checked by SDS-PAGE. The HuA5.2 antibody had a size of about 100Kd and after reduction was about 50Kd, and the sizes of the protein bands were all correct as shown in FIG. 6.
Example 8 comparison of the affinity difference between HuA5.2 and A5 binding to GPC3
GPC3 antigen protein (Sino Biological, Cat:10088-H02H2) was coated on the bottom of ELISA plates, and HuA5.2 and chicken anti-A5 were added to the plate at a concentration of 20nM in the first well at 1: 3, adding the mixture into the wells after gradient dilution, then adding the HRP-goat anti-human antibody, and finally developing. As shown in fig. 7, the EC50 value of hua5.2 was 0.08628, whereas the EC50 value of a5 was 0.0961, indicating that the binding ability of hua5.2 to GPC3 protein was not reduced, even higher affinity, than that of a5.
Example 9ELIspot detection of immunogenicity of HuA5.2 antibodies
Human IFN-. gamma.precoated ELISPOT kit was purchased from Dake, Inc., Cat # 211005(strips), and the protocol was described in the kit. Briefly, antibodies and PNMC are incubated on an ELISPOT plate, IFN-gamma secreted by the PBMC is captured by IFN-gamma antibodies which are fixed on the bottom of the plate, then spots are formed on the bottom of the plate through color development, and the immunogenicity of the HuA5.2 is judged by counting the number of the spots. The larger the number of spots, the more IFN- γ secreted by PBMC, the stronger the immune response to the protein tested, i.e.the greater the immunogenicity, and vice versa. As shown in fig. 8, the number of spots for hua5.2 was significantly less than a5, indicating that hua5.2 stimulated T cells to produce less IFN- γ and was less immunogenic.
Sequence listing
<110> university of agriculture in Huazhong
<120> humanized phosphatidylinositolglycan 3 monoclonal antibody and application thereof
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Thr Cys Ser Gly Asp Gly Ser Tyr Ala Tyr Trp Tyr
20 25 30
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Asp Asn Asp
35 40 45
Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly
50 55 60
Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Gln Leu Thr Val Leu
100 105
<210> 3
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 4
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 5
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 107
<212> PRT
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<400> 6
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 7
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 8
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ser Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 9
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 107
<212> PRT
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<400> 10
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Asp Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 11
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 12
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 13
<211> 126
<212> PRT
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<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 107
<212> PRT
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<400> 14
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Tyr Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 15
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 17
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 19
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 20
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly
50 55 60
Ser Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala
65 70 75 80
Asp Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 21
<211> 126
<212> PRT
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<400> 21
Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly
1 5 10 15
Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Asn Arg Tyr
20 25 30
Cys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Asp Ser Asp Ser Gly Gly Thr Asp Tyr Gly Ala Ala Val
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg
65 70 75 80
Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Lys Ser Ala Tyr Gly Gly Trp Cys Gly Ser Arg Val Ala Pro Trp
100 105 110
Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser
115 120 125
<210> 22
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gln Ala Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Thr Cys Ser Gly Gly Gly Ser Tyr Gly Trp Phe Gln
20 25 30
Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Asp Asn Asp
35 40 45
Lys Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly
50 55 60
Ser Thr Gly Thr Leu Thr Ile Thr Gly Val Arg Ala Glu Asp Glu Ala
65 70 75 80
Val Tyr Tyr Cys Gly Ser Ser Glu Asn Ser Tyr Val Gly His Val Ala
85 90 95
Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu
100 105

Claims (13)

1. A humanized monoclonal antibody to glypican 3, characterized in that the antibody comprises a heavy chain variable region and a light chain variable region, the framework region of the heavy chain variable region: the HFR1 amino acid sequence is shown as 1-25 of SEQ ID NO. 3, the HFR2 amino acid sequence is shown as 35-49 of SEQ ID NO. 3 or 35-49 of SEQ ID NO. 19, the HFR3 amino acid sequence is shown as 67-98 of SEQ ID NO. 3, 67-98 of SEQ ID NO. 15, or any one of 67-98 of SEQ ID NO. 17, the HFR4 amino acid sequence is shown as 116-126 of SEQ ID NO. 3, and the framework region of the light chain variable region is: the LFR1 amino acid sequence is shown as 1-22 bits of SEQ ID NO. 4, the LFR2 amino acid sequence is shown as 30-45 bits of SEQ ID NO. 4 or 30-45 bits of SEQ ID NO. 14, the LFR3 amino acid sequence is shown as 53-84 bits of SEQ ID NO. 4, 53-84 bits of SEQ ID NO. 6, 53-84 bits of SEQ ID NO. 8, 53-84 bits of SEQ ID NO. 10, or any one of 53-84 bits of SEQ ID NO. 12, and the LFR4 amino acid sequence is shown as 98-107 bits of SEQ ID NO. 4.
2. The humanized glypican 3 monoclonal antibody as claimed in claim 1, characterized in that:
(1) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 3; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO. 4;
(2) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 5; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 6;
(3) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 7; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 8;
(4) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 9; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 10;
(5) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 11; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 12;
(6) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 13; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO. 14;
(7) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 15; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 16;
(8) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO: 17; the variable region of the light chain of the antibody has a framework region shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO 18;
(9) the heavy chain variable region of the antibody has a framework region shown by amino acid residues 1-25, 35-49, 67-98 and 116-126 of SEQ ID NO 19; the variable region of the light chain of the antibody has framework regions shown by amino acid residues 1-22, 30-45, 53-84 and 98-107 of SEQ ID NO: 20.
3. The humanized glypican 3 monoclonal antibody as claimed in claim 1, characterized in that:
1) the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 3, and the light chain variable amino acid sequence is shown as SEQ ID NO. 4.
2) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO.5, and the light chain variable amino acid sequence is shown as SEQ ID NO. 6.
3) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 7, and the light chain variable amino acid sequence is shown as SEQ ID NO. 8.
4) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 9, and the light chain variable amino acid sequence is shown as SEQ ID NO. 10.
5) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 11, and the light chain variable amino acid sequence is shown as SEQ ID NO. 12.
6) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 13, and the light chain variable amino acid sequence is shown as SEQ ID NO. 14.
7) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 15, and the light chain variable amino acid sequence is shown as SEQ ID NO. 16.
8) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 17, and the light chain variable amino acid sequence is shown as SEQ ID NO. 18.
9) The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 19, and the light chain variable amino acid sequence is shown as SEQ ID NO. 20.
4. The monoclonal antibody according to claim 1 or 2, characterized in that the heavy chain variable region of the antibody has complementarity determining regions represented by amino acid residues 26-34, 50-66 and 99-115 of SEQ ID NO 3; the variable region of the light chain of the antibody has complementarity determining regions shown by amino acid residues 23-29, 46-82 and 85-97 of SEQ ID NO. 4.
5. The humanized glypican 3 monoclonal antibody as claimed in any one of claims 1 to 4, characterized in that it further comprises a tag sequence for aiding expression and/or purification.
6. An immunoconjugate characterized in that it comprises a monoclonal antibody of humanized glypican 3 according to any one of claims 1 to 5 and an effector molecule.
7. The immunoconjugate according to claim 6, characterized in that the effector molecule is a toxin, a drug or a detectable label.
8. The immunoconjugate according to claim 7, characterized in that the toxin is a protein with cytotoxic or anti-tumor activity comprising abrin or a variant thereof, ricin or a variant thereof, pseudomonas exotoxin or a variant thereof, diphtheria toxin or a variant thereof, botulinum toxin or a variant thereof; the drug is a compound having cytotoxic or antitumor activity, including Monomethylauristatin E, Monomethylauristatin F, Pyrolobe diazepine dimer, N2'-deacetyl-N2' - (3-Mercapto-1-oxopropyl) -Maytansine; the detectable marker is a substance that can be detected by an isotope analyzer, a microplate reader, a bioluminescent detector, a chemiluminescent detector, an electrochemiluminescent detector, a fluorescence analyzer, or visualized by the naked eye.
9. A polynucleotide, characterized in that it encodes a monoclonal antibody of humanized glypican 3 according to any one of claims 1 to 5, or an immunoconjugate according to any one of claims 6 to 8.
10. A vector comprising the polynucleotide of claim 9.
11. A genetically engineered host cell comprising the vector of claim 10, or having the polynucleotide of claim 8 integrated into its genome.
12. A pharmaceutical composition comprising one or more of a humanized glypican 3 monoclonal antibody according to any one of claims 1 to 5, an immunoconjugate according to any one of claims 6 to 8, a polynucleotide according to claim 9, a vector according to claim 10 or a genetically engineered host cell according to claim 11.
13. Use of a humanized glypican 3 monoclonal antibody according to any one of claims 1 to 5, or the immunoconjugate according to any one of claims 6 to 8, the polynucleotide according to claim 9, the vector according to claim 10 or the genetically engineered host cell according to claim 11 for the preparation of a therapeutic drug or a diagnostic agent for autoimmune diseases, viral infections or cancer.
CN202011158008.2A 2020-10-26 2020-10-26 Humanized phosphatidylinositolglycan 3 monoclonal antibody and application thereof Pending CN114478779A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596985A (en) * 2011-04-19 2014-02-19 美国政府(由卫生和人类服务部的部长所代表) Human monoclonal antibodies specific for glypican-3 and use thereof
US20150147330A1 (en) * 2012-06-01 2015-05-28 The United States of America,as represented by the Secretary,Department of Health and Human Service High-affinity monoclonal antibodies to glypican-3 and use thereof
CN110627904A (en) * 2019-10-31 2019-12-31 南京蓝盾生物科技有限公司 Anti-human GPC3 monoclonal antibody

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596985A (en) * 2011-04-19 2014-02-19 美国政府(由卫生和人类服务部的部长所代表) Human monoclonal antibodies specific for glypican-3 and use thereof
US20150147330A1 (en) * 2012-06-01 2015-05-28 The United States of America,as represented by the Secretary,Department of Health and Human Service High-affinity monoclonal antibodies to glypican-3 and use thereof
CN110627904A (en) * 2019-10-31 2019-12-31 南京蓝盾生物科技有限公司 Anti-human GPC3 monoclonal antibody

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